FoxO transcription factors control aging in invertebrates, and FoxO polymorphisms are associated with extreme longevity in multiple independent cohorts of long-lived humans. The phenotypes of conditional FoxO knockout mice suggest that FoxO transcription factors may also be involved in the pathogenesis of cancer, Type 2 diabetes, and osteoporosis. Thus, understanding how FoxO transcription factors are regulated is expected to illuminate conserved mechanisms underlying normal aging while laying a foundation for advances in the prevention, diagnosis, and treatment of common human diseases. Major gaps in the understanding of how FoxO transcription factors are regulated in lifespan control are highlighted by the finding that the C. elegans serum- and glucocorticoid-induced kinase ortholog SGK-1 controls lifespan by promoting the activity of the FoxO transcription factor DAF-16. This is at odds with current paradigms of FoxO inhibition by Sgk and underscores the need for additional investigation into mechanisms of FoxO regulation. The long-term goal is to understand how upstream signaling inputs regulate FoxO transcription factor activity to control lifespan. The objective of this proposal is to elucidate the molecular basis for the DAF-16/FoxO-dependent lifespan extension observed in C. elegans sgk-1 gain-of-function mutants. C. elegans remains an excellent system for studying FoxO regulation in lifespan control. It is a tractable system for assessing the impact of experimental perturbations on organismal lifespan; furthermore, the major FoxO regulatory inputs that impact lifespan, as well as three major regulatory phosphorylation sites on FoxO, are conserved between C. elegans and humans. The central hypothesis is that the DAF-16/FoxO N-terminal phosphorylation site is a conserved switch that determines the functional consequences of DAF-16/FoxO C-terminal site phosphorylation by SGK-1. The rationale for the proposed research is that an understanding of how SGK-1 promotes longevity by regulating DAF-16/FoxO activity is a first step toward understanding how FoxO transcription factor regulation influences aging and the pathogenesis of aging-related diseases in humans. Two specific aims are proposed to accomplish the objective. In Aim 1, the hypothesis that SGK-1 controls lifespan by phosphorylating DAF-16/FoxO at the conserved C-terminal site will be tested. In Aim 2, the hypothesis that SGK-1 activates DAF-16/FoxO when the conserved N-terminal site is not phosphorylated but inhibits DAF-16/FoxO by promoting its translocation to the cytoplasm when the conserved N-terminal site is phosphorylated will be tested. The research proposed here is innovative because it will test a model of FoxO regulation by Sgk that differs substantively from the current paradigm of Sgk as a FoxO inhibitor. We expect the proposed research to yield novel insights into the in vivo roles of conserved FoxO phosphorylation events in lifespan control. This contribution will be significant because it is expected to lead to the development of new pharmacologic strategies to promote healthy aging and combat common human diseases associated with aging by modulating FoxO transcription factor activity. PUBLIC HEALTH RELEVANCE: This project is relevant to public health because the elucidation of evolutionarily conserved mechanisms of FoxO transcription factor regulation in lifespan control is expected to illuminate the pathogenesis of prevalent aging-related diseases such as cancer, Type 2 diabetes, and osteoporosis. Thus, the proposed research is relevant to the mission of NIH in that a better understanding of the aging process will improve human health by engendering the development of new strategies to prevent, diagnose, and treat common human diseases associated with aging.